reversible inactivation of the dorsal nucleus of the ... · reversible inactivation of the dorsal...

Reversible Inactivation of the Dorsal Nucleus of the LateralLemniscus Reveals Its Role in the Processing of Multiple SoundSources in the Inferior Colliculus of Bats

R. Michael Burger and George D. Pollak

Section of Neurobiology, University of Texas, Austin, Texas 78712

Neurons in the inferior colliculus (IC) that are excited by one earand inhibited by the other [excitatory—inhibitory (EI) neurons]can code interaural intensity disparities (IIDs), the cues animalsuse to localize high frequencies. Although EI properties are firstformed in a lower nucleus and imposed on some IC cells via anexcitatory projection, many other EI neurons are formed denovo in the IC. By reversibly inactivating the dorsal nucleus ofthe lateral lemniscus (DNLL) in Mexican free-tailed bats withkynurenic acid, we show that the EI properties of many IC cellsare formed de novo via an inhibitory projection from the DNLLon the opposite side. We also show that signals excitatory tothe IC evoke an inhibition in the opposite DNLL that persists fortens of milliseconds after the signal has ended. During thatperiod, strongly suppressed EI cells in the IC are deprived ofinhibition from the DNLL and respond to binaural signals asweakly inhibited or monaural cells. By relieving inhibition at the

IC, we show that an initial binaural signal essentially reconfig-ures the circuit and thereby allows IC cells to respond to trailingbinaural signals that were inhibitory when presented alone.Thus, DNLL innervation creates a property in the IC that is notpossessed by lower neurons or by collicular EI neurons that arenot innervated by the DNLL. That property is a change inresponsiveness to binaural signals, a change dependent on thereception of an earlier sound. These features suggest that thecircuitry linking the DNLL with the opposite central nucleus ofthe IC is important for the processing of IIDs that change overtime, such as the IIDs generated by moving stimuli or bymultiple sound sources that emanate from different regions ofspace.

Key words: GABA; persistent inhibition; precedence effect;inferior colliculus; sound localization; dorsal nucleus of laterallemniscus

The projections from the vast majority of lower auditory nucleiconverge at a common destination in the central nucleus of theinferior colliculus (ICc) (for review, see Aitkin, 1986; Oliver andHuerta, 1992). This large convergence of inputs suggests thatsubstantial transformations occur in the ICc; yet the responseproperties of ICc neurons appear to be similar to those of thelower nuclei from which they receive their innervation. An exam-ple is excitatory–inhibitory (EI) neurons in the ICc, neurons thatare excited by one ear and inhibited by the other ear. Theseneurons are sensitive to interaural intensity disparities (IIDs), thecues animals use to localize high-frequency sounds (Erulkar,1972; Mills, 1972). EI neurons are initially formed in the lateralsuperior olive (LSO) (Boudreau and Tsuchitani, 1968; Finlaysonand Caspary, 1991). They are also the dominant type in the dorsalnucleus of the lateral lemniscus (DNLL), a nucleus the neuronsof which are almost exclusively GABAergic (Brugge et al., 1970;Adams and Mugniani, 1984; Covey, 1993; Yang and Pollak,1994a,b,c; Winer et al., 1995). The DNLL, like the ICc, is one ofthe principal targets of both the ipsilateral and contralateral LSOs(Glendenning et al., 1981; Ross et al., 1988; Shneiderman et al.,

1988, 1999; Yang et al., 1996). The DNLL, in turn, sends strongGABAergic projections bilaterally to the ICc (Ross et al., 1988;Shneiderman et al., 1988, 1999; Gonzalez-Hernandez et al., 1996;Kelly et al., 1998). Thus, EI cells of the ICc are strongly inner-vated by both LSOs and DNLLs, the lower nuclei the neuronalpopulations of which are EI (Fig. 1A).

The projections from the contralateral LSO to both the DNLLand ICc are excitatory, and it is via these crossed projections thatthe EI properties of the LSO are imposed on the DNLL and ICc(Fig. 1B,C) (Saint Marie et al., 1989; Saint Marie and Baker,1990; Glendenning et al., 1992; Park and Pollak, 1993, 1994).However, studies over the past several years have shown thatalthough the EI properties of some ICc neurons are derived frominnervation by the LSO, the EI properties of many ICc cells areeither modified substantially or even formed de novo in the ICc(Faingold et al., 1991; Li and Kelly, 1992; Vater et al., 1992;Faingold et al., 1993; Park and Pollak, 1993, 1994). In this regard,the GABAergic projections from the DNLL to the opposite ICcplay critical roles (Fig. 1C,D).

What is unclear is what functional dividend derives frommodifying or forming EI properties de novo in the ICc, becauseEI cells are already formed in the LSO. One hypothesis proposesthat the reception of a first signal reconfigures the circuit byfunctionally inactivating the DNLL for a period of time. Duringthat period, EI cells in the IC are deprived of their inhibitoryinputs from the DNLL and are temporarily transformed fromstrongly inhibited into weakly inhibited EI or even monaural cells(Yang and Pollak, 1994c; Pollak, 1997). In short, there would bea change in the responsiveness of ICc cells to binaural signals, achange dependent on the reception of an earlier sound that

Received Jan. 17, 2001; revised April 9, 2001; accepted April 9, 2001.This work was supported by Grant RO1 DC 00268-16 from the the National

Institute on Deafness and Other Communicative Disorders, National Institutes ofHealth. We thank Eric Bauer, Jane Lubischer, Jeff Wenstrup, Achim Klug, WaltWilczynski, and David Ryugo for their comments and suggestions, Linslee Luke forher assistance with the histology, and Carl Resler for technical support.

Correspondence should be addressed to Dr. George D. Pollak at the aboveaddress. E-mail: [email protected].

R. M. Burger’s present address: Virginia Merrill Bloedel Hearing ResearchCenter, University of Washington, Seattle, WA 98195-7923.Copyright © 2001 Society for Neuroscience 0270-6474/01/214830-14$15.00/0

The Journal of Neuroscience, July 1, 2001, 21(13):4830–4843

inactivates the DNLL. Thus, at least one consequence of DNLLprojections to the ICc is to influence the processing of multiplesound sources that originate from different regions of space.

Here we provide evidence supporting this hypothesis withrecordings from the auditory system of Mexican free-tailed bats,where we block GABAergic inhibition at the DNLL and ICc aswell as reversibly inactivate the DNLL while recording from ICcneurons. We then discuss the functional relevance of these fea-tures for sound localization and suggest that DNLL inhibition ofthe ICc may be one of the neural mechanisms that underlie theprecedence effect.

MATERIALS AND METHODSSurgical procedure. Mexican free-tailed bats, Tadarida brasilensis mexicana,were used in this study. We used these animals because their brainstemauditory nuclei are fundamentally similar to those of less specializedmammals, but they are greatly hypertrophied (Grothe et al., 1994; Park etal., 1996; Grothe and Park, 1998). The relatively large size makes nuclei,such as the IC, readily accessible, whereas the small absolute size allowsthem to be reversibly inactivated by iontophoresing drugs.

Each animal was anesthetized with methoxyflurane inhalation (Meto-

fane; Pitman-Moore, Inc.) and the neuroleptic Innovar-Vet (Fentanyland Droperidol; 0.02 mg/gm of body weight; Pitman-Moore, Inc.), in-jected intraperitoneally. The hair on the head was removed with adepilatory, and the head was secured in a head holder with a bite bar.The muscles and skin overlying the skull were reflected, and lidocaine(Elkins-Sinn, Cherry Hill, NJ) was applied topically to all open wounds.The surface of the skull was cleared of tissue, and a foundation layer ofcyanoacrylate and small glass beads was placed on the surface. The IC isgreatly hypertrophied and is so large that it protrudes between the cortexand the cerebellum. Both ICs are clearly visible through the thin brain-case as two prominent structures framed by suture lines. By the use ofvisible landmarks formed by the ICs and the suture lines, a small openingin the skull was made over the left IC with a scalpel blade. In experimentsin which we recorded from the DNLL, a small opening was made overthe right IC, and in experiments in which we inactivated the DNLL whilerecording from the ICc, small openings were made over each IC.

The bat was then transferred to a heated recording chamber, where itwas placed in a restraining cushion constructed of foam molded to theanimal’s body. The restraining cushion was attached to a platformmounted on a custom-made stereotaxic instrument (Schuller et al., 1986).A small metal rod was cemented to the foundation layer on the skull andthen attached to a bar mounted on the stereotaxic instrument to ensurea uniform positioning of the head. A ground electrode was placedbetween the reflected muscle and the skin. Recordings were begun afterthe bats recovered from the anesthetic. The bats typically lie quietly inthe restraining cushion and show no signs of pain or discomfort. Supple-mentary doses of the neuroleptic were given if the bat struggled orotherwise appeared in discomfort.

After the animal was fixed in the stereotaxic instrument, the electrodewas positioned over the right IC while being viewed with an operatingmicroscope. The electrode was advanced to a depth of 300 mm to ensurethat recordings were obtained from neurons in the central nucleus of theinferior colliculus. The electrode was subsequently advanced from outsideof the experimental chamber with a piezoelectric microdrive (Burleigh7121W). For recordings from the DNLL, the instrument in which the batwas held was first rotated and adjusted to maximize the extent of theDNLL that would be encountered by the electrode penetration. By the useof visual landmarks, the electrode was then positioned on the surface of theIC so that it would enter the DNLL at a depth of ;1600 mm. As with ICcrecordings, the electrode was subsequently advanced from outside of theexperimental chamber with a piezoelectric microdrive.

Several criteria were used to determine when the electrode had left theICc and entered the DNLL. As the electrode was advanced through theICc, there was an abrupt change in the best frequency (BF, the frequencyto which the cluster or single unit was most sensitive) of the backgroundactivity at a depth of ;1600 mm. This change signaled that the electrodehad left the ICc and entered the DNLL. For the next 200–300 mm, boththe multiunit activity and single units encountered displayed prominentsustained activity in response to contralateral tone bursts, and thisactivity was strongly suppressed when tone bursts were presented simul-taneously to the ipsilateral (inhibitory) ear. A second abrupt change inBF and a change from binaural activity to monaural activity that wasinfluenced only by sound to the contralateral ear signaled when theelectrode left the DNLL and entered the intermediate nucleus of thelateral lemniscus. To ensure that these changes in BF, as well as dis-charge patterns and binaural properties, indicate DNLL location, in 16experiments electrode location was verified with small iontophoreticinjections (2–5 nA for 20–60 sec) of rhodamine-conjugated dextran(Molecular Probes, Eugene, OR). These response features have provento be reliable indicators in previous studies of the mustache bat’s DNLL(Markovitz and Pollak, 1993, 1994; Yang and Pollak, 1994a,b,c).

Electrodes. Both single-barrel glass pipettes filled with 1 M NaCl and2% fast green, pH 7.4, and “piggy back” multibarrel micropipettes(Havey and Caspary, 1980) were used. Fast green was used to enhancethe visibility of the electrode. The recording electrodes, used eithersingly or when mounted on multibarrel electrodes, were constructed fromcapillary glass that was pulled and blunted to a tip diameter of 1–2 mmunder microscopic observation. Multibarrel electrodes were pulled froma five-barrel blank (A-M Systems) and blunted to 15–20 mm. A single-barrel pipette was then attached to the five-barrel pipette and glued withcyanoacrylate so that the tip of the single-barrel pipette protruded 10–15mm from the blunted tip of the five-barrel pipette. The single-barrelmicropipette was used for recording single-unit activity and was filledwith buffered 1 M NaCl and 2% fast green, pH 7.4. One barrel of thefive-barrel pipette was the balancing barrel and was filled with buffered 1

Figure 1. Schematic diagrams showing some principal connections fromlower nuclei to EI neurons in the ICc ( A) and the various ways that EIproperties can be formed by subsets of those projections (B–D). A, TheDNLL, shown in black, is a purely GABAergic nucleus that providesstrong inhibitory projections to both the ipsilateral and contralateral ICc.Excitatory projections are shown as solid lines, and inhibitory projectionsare dashed lines. B, Excitatory projections from LSO to the contralateralICc are shown. It is via this pathway that the excitatory–inhibitoryproperties first formed in the LSO can be imposed on their targets in theICc. C, This circuit shows how EI properties can be formed de novo in theICc. Stimulation of the ear contralateral to the ICc drives a lowermonaural nucleus, shown generically here as the cochlear nucleus, whichprovides the excitation to the ICc. Stimulation of the ear ipsilateral to theICc excites the DNLL, which then provides the inhibition that suppressesthe contralaterally evoked excitation in the ICc. D, This circuit shows howEI properties, first formed in the LSO, can be modified in the ICc via theconvergence of LSO and DNLL projections. The net effect of thisconvergence is to create EI cells in the ICc that are suppressed by lowerintensities at the ipsilateral ear than they would be if they received onlythe LSO projection. excit, Excitatory; inhib, inhibitory; MNTB, medialnucleus of the trapezoid body.

Burger and Pollak • Reversible Inactivation of DNLL Impacts Inferior Colliculus J. Neurosci., July 1, 2001, 21(13):4830–4843 4831

M NaCl and 2% fast green. The other barrels were filled with varioussubstances that depended on the experiment. With ICc recordings, twoto four barrels were filled with solutions of bicuculline methiodide (10mM in 0.165 M NaCl, pH 3.0; Sigma, St. Louis, MO), an antagonist ofGABAA receptors (Borman, 1988), and in some experiments one barrelwas also filled with glutamic and aspartic acid (500 mM each in dH2O, pH9–10). When recording from the DNLL, two barrels were filled with acocktail of bicuculline methiodide and the glycine receptor antagoniststrychnine HCl (Cooper et al., 1982) (both were 10 mM in 0.165 M NaCl,pH 3.0; Sigma). The two other barrels were filled with glutamic andaspartic acid (500 mM each in dH2O, pH 9–10). As explained in Results,because neither ICc nor DNLL cells were spontaneously active, glutamicand aspartic acid were used to generate background activity againstwhich inhibition evoked by stimulation of the ipsilateral ear could beobserved. For DNLL inactivation experiments, all drug barrels werefilled with a solution of kynurenic acid (75 mM in 0.165 M NaCl, pH 9–10;Sigma), a broad-spectrum antagonist of glutamatergic receptors (Col-lingridge and Lester, 1989). Drugs were retained in the electrode with a15–20 nA current of opposite polarity. The drug and balancing barrelswere connected via silver–silver chloride wires to a six-channel micro-iontophoresis constant-current generator (Medical Systems NeurophoreBH-2) that was used to generate and monitor ejection and retentioncurrents. The sum channel was used to balance current in the drugbarrels and reduce current effects. The recording barrel was connected bya silver–silver chloride wire to a Dagan AC amplifier (model 2400).

Acoustic stimuli and data acquisition. Sine waves from a Wavetekfunction generator (model 136) were shaped into tone bursts with acustom-made analog switch. Tone burst frequency was monitored by afrequency counter. Tone bursts, as well as frequency-modulated (FM)sweeps, were also generated digitally by a Power Macintosh 7300. FMsweeps were always digitally generated and could have any desiredduration as well as starting and terminal frequency. All stimuli, whethergenerated digitally or by the function generator, had 0.2 msec rise andfall times and were presented four times per second. The computer couldgenerate either one or two signals (tone bursts or FM sweeps), withinterpulse intervals selected by the investigator. The interpulse intervalwill be referred to simply as the “interval” and indicates the time fromthe end of the first signal to the beginning of the second or trailing signal.In addition, each signal could be presented either monaurally or binau-rally. A Power Macintosh 7300 computer was used to control stimulusparameters via connections to a real time clock and a pair of digitalattenuators (Wilsonics, model PATT) through a 24-bit digital interfaceNuBus card (National Instruments DIO-24) and a custom-made digitaldistributor. The output of each independently controlled channel of theattenuator was sent to two 1/4 inch Bruel and Kjaer (B&K) microphonesbiased with 200 V of DC and driven as speakers. The frequency responseof each speaker was measured with another B&K microphone, the outputof which was fed to a sound-level meter (B&K, model 2608) thatmeasured sound pressure level in decibels (0.0002 dynes/cm 2). At thestart of each experiment, speakers, with the protective grid attached,were inserted into the funnels formed by the bat’s pinnas and were posi-tioned adjacent to the external auditory meatus. The pinnas were foldedonto the housing of the microphones and wrapped with Scotch tape. Thebinaural cross-talk with this arrangement was attenuated by 35–40 dB.

Spikes were fed to a window discriminator and then to a PowerMacintosh 7300 computer controlled by a custom-made real time clock.Raster displays, peristimulus time histograms, and rate-level functionswere generated and graphically displayed. Unless otherwise noted, eachraster display was generated from the discharges evoked by 20 presenta-tions of a signal at a fixed intensity.

After a unit was isolated, its BF, threshold at BF, and binaural typewere determined audiovisually. Binaural properties were determined bypresenting a BF tone burst 10–30 dB above threshold, first to thecontralateral (excitatory) ear, then to the ipsilateral (inhibitory) ear, andthen to both ears simultaneously. When presented simultaneously, ipsi-lateral intensities, ranging from 20–30 dB below to 20–30 dB above theintensity of the contralateral ear, were presented to determine theipsilateral intensities that suppressed contralaterally evoked discharges.

Spike counts evoked by increasing ipsilateral intensities of binauralsignals were normalized in terms of the percentage change from themaximum response. Maximum response was the spike count evoked bythe contralateral signal alone or the spike count evoked by a binauralsignal with the lowest ipsilateral intensity, an ipsilateral intensity that wasalways below threshold for evoking inhibition.

In many ICc recordings we presented an initial and a trailing signal to

evaluate the effects of an initial signal on the binaural properties evokedby trailing signals. In all of these experiments, the first signal was alwaysa 5–10 msec FM signal that swept downward by 10–30 kHz. The initialand terminal frequencies were adjusted so that the frequencies sweptthrough the BF of the neuron. The binaural trailing signals were eitherFM sweeps, identical to the first FM signal, or BF tone bursts. We did notuse tone bursts as the initial signals because in the vast majority of ICcneurons, BF tone bursts at the contralateral (excitatory) ear evoke anexcitation followed by a long-lasting inhibition (Klug et al., 1999; Baueret al., 2000). The inhibition prevents ICc neurons from responding totrailing signals and creates long recovery times. In contrast, FM sweepsproduce such inhibition much less frequently, and most ICc neuronsrecover within a few milliseconds (G. Pollak et al., 1977; G. D. Pollak etal., 1977).

After recording the responses of a cell to these signals, pharmacolog-ical agents were iontophoretically applied, and the responses to the samesignals were recorded again. During application of bicuculline or thecocktail of bicuculline and strychnine, rate-level functions were moni-tored while ejection currents of from 10 to 60 nA were applied. For eachejection current, rate-level functions were obtained until the rate-levelfunction stabilized. After the responses were stable, the complement oftone bursts and FM signals was presented again, and the same responsefeatures were obtained for comparison with those obtained before theapplication of drugs. The ejection current was then switched off, and thecell was allowed to recover. Recovery was complete when both the shapeof the rate-level function returned to its predrug form and the maximumspike count returned to the predrug value. In many cases, however,contact with the unit was lost before recovery was complete. Becauserecovery times were usually 10–20 min, when neurons were lost beforerecovery was attained, we allowed at least 30 min before searching foranother neuron.

Procedures for reversibly inactivating the DNLL. We opted to inactivatethe DNLL with iontophoretic application of kynurenic acid rather thanwith pressure. The main reason was that we wanted to minimize anypotential tissue damage caused by the mechanical deformations thatresult from the relatively large volumes of liquid injected with pressure.Thus, we wanted to inactivate repeatedly the DNLL and be confidentthat the previous inactivation had not caused damage to the DNLL.

We first determined the dimensions of the DNLL. The DNLL stainsintensely with cytochrome oxidase and stands out clearly from otherstructures (Markovitz and Pollak, 1994). We processed four brains forcytochrome oxidase and determined the largest rostrocaudal, mediolat-eral, and dorsoventral extent of the DNLL. On average, the dimensionswere as follows: rostrocaudal 554 mm, mediolateral 524 mm, and dorso-ventral 282 mm. The histological and other procedures used to determinethese dimensions are described below. Assuming that kynurenic acidspreads spherically from the injection site, we calculated that the requiredradius of spread had to be ;275 mm to inactivate most of the DNLL.

We next obtained an estimate of how far kynurenic acid spreads fromthe ejection barrel. This was accomplished by making multibarrel elec-trodes in which the tips of the recording electrodes were 100–275 mmbeyond the tips of the multibarrel electrodes from which the kynurenicacid was ejected. We then recorded from 11 single units in the ICc of fourbats. The ICc was used because it was easily accessible. With appropriateadjustments of ejection currents, discharges could be completely sup-pressed within 1–3 min, even when the recording electrode was 275 mmdistant from the ejection electrodes, and discharges recovered within 3–5min (e.g., Fig. 2 A). Reapplication of kynurenic acid again completelysuppressed discharges, and responsiveness again recovered within 3–5min (Fig. 2 B). The degree of suppression, the time it took to achievesuppression, and the time for recovery were all dosage dependent (e.g.,Fig. 2 B). These results provide confidence that iontophoretic applicationof kynurenic acid inactivated much of the DNLL and may even havespread slightly beyond the dorsoventral limits of the DNLL. Further-more, it shows that inactivated neurons recover and suggests that re-peated applications of kynurenic acid produce no tissue damage.

For DNLL inactivation experiments, the location of the DNLL wasinitially identified using a single-barrel electrode, using the criteria de-scribed above. After the DNLL was located with the single-barrelpipette, a multibarrel electrode was put in its place and was advancedwhile multiunit responses were monitored to ensure that the multibarrelelectrode was in the DNLL. The inactivating electrode was positionedmidway along the dorsoventral extent of the DNLL.

For each neuron we determined the percentage relief from inhibitionbecause of DNLL inactivation. Spike counts evoked by binaural signals

4832 J. Neurosci., July 1, 2001, 21(13):4830–4843 Burger and Pollak • Reversible Inactivation of DNLL Impacts Inferior Colliculus

before inactivation were normalized in terms of the percentage changefrom the maximum response. Normalized spike counts were then deter-mined during DNLL inactivation, and the difference between the twofunctions is given as the percentage relief from inhibition. For example,if contralaterally evoked discharges were completely suppressed at agiven ipsilateral intensity, the suppression would be 100%. If, at the sameipsilateral intensity, the suppression was 60% during DNLL inactivation,the percentage relief at that ipsilateral intensity is 40%. Our criterion for“relief” was at least a 20% relief of the suppression produced at thoseipsilateral intensities that, before inactivation, suppressed contralaterallyevoked discharges by at least 50%.

Histolog ical procedures. For localizing electrode locations in theDNLL after injections of rhodamine conjugated to dextran, animalswere allowed to survive for 1 d. They then were deeply anesthetized withMetofane and perfused intracardially with 0.1 M phosphate buffer fol-lowed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brains wereremoved, post-fixed for 4–24 hr, and then kept overnight in 30% sucrose.Sections were cut on a freezing microtome and mounted in FlouromountG (Southern Biotechnology, Birmingham, AL). Sections were viewed

under a fluorescent microscope, and images of the DNLL were capturedusing a video frame grabber for archival purposes.

To determine the dimensions of the DNLL, bats were perfused asdescribed above, and the brains were blocked and post-fixed for 24 hrbefore submersion in 30% sucrose. Sections (40 or 50 mm) were madethrough the portion of the brain containing the DNLL. Two brains werecut in the sagittal plane, and two others were cut in the transverse plane.Sections were processed using procedures described by Wong-Riley(1979). Sections were mounted and incubated in the dark at 37°C for 1–2hr until brown reaction product was observed in the tissue. The incuba-tion medium contained 50 mg of diaminobenzidine tetrahydrochloride(Sigma) in 90 ml of 0.1 M phosphate buffer, pH 7.4, 15 mg of cytochromeC type III (Sigma), and sucrose. Sections were imaged with a video framegrabber, and areas were computed with NIH Image software.

RESULTSWe report on 150 EI cells from the ICc and 113 EI neurons fromthe DNLL of Mexican free-tailed bats. The BFs, the frequenciesto which each neuron was most sensitive, of the ICc neuronsranged from 19 to 36 kHz, and the BFs of the DNLL cells rangedfrom 18 to 79 kHz. The EI property of each neuron was deter-mined by presenting a sound to the excitatory ear at a fixedintensity that was 10–30 dB above threshold and then presentingthe same sound to the inhibitory ear at progressively increasingintensities. Because the intensity at the excitatory ear was fixed,each change in intensity at the inhibitory ear produced a differentIID. The more intense inhibitory signals strongly suppresseddischarges evoked by excitatory signals in most ICc cells. Thedegree of maximal suppression varied among the ICc population,although the maximal spike suppression in most cells (71%) wasat least 90%. The average suppression was 91% and ranged from30 to 100%.

The focus of this study was on the influences of the GABAergicprojections from the DNLL to the opposite ICc and the func-tional significance of those influences. Below we first discuss somefeatures of inhibition in DNLL neurons. We then show that manyICc neurons are influenced by GABAergic innervation from theopposite DNLL, and in the final sections we show the functionalsignificance of those DNLL projections on the processing of IIDsin the ICc.

Persistent inhibition is a key feature of DNLL neuronsPrevious studies in mustache bats have shown that inhibition inthe DNLL, evoked by stimulation of the ear ipsilateral to it, ispersistent, in that the inhibition often lasts for many millisecondsafter the end of the signal that evoked it (Yang and Pollak,1994a,b, 1998). The duration of the inhibitory persistence, whichcan be as long as 80 msec, changes little with stimulus duration butincreases with intensity and typically reaches a maximum dura-tion 20–40 dB above the threshold for inhibition (Yang andPollak, 1994a,c). Here we show that persistent inhibition is also aprominent feature of DNLL neurons in Mexican free-tailed bats.In a later section we demonstrate the functional impact thatpersistent inhibition in the DNLL has on many cells in the ICc.

We tested 50 DNLL cells in Mexican free-tailed bats forpersistent inhibition and illustrate this inhibition with the DNLLneuron in Figure 3. Figure 3A shows that the neuron was drivenby tone bursts presented to the contralateral (excitatory) ear.Stimulation of the ipsilateral (inhibitory) ear evoked no responses(data not shown). To show that ipsilateral stimulation evoked aninhibition in the DNLL, we created background discharges byiontophoresing the excitatory transmitters glutamate and aspar-tate (Fig. 3B). Ipsilateral (inhibitory) tone bursts, at 40 dB soundpressure level (SPL) and 10 msec in duration, produced a gap inthe glutamate-evoked discharges that lasted for 70 msec (Fig. 3C).

Figure 2. Inactivation of single-unit activity by iontophoresis ofkynurenic acid. Tone bursts were presented once every 5 sec. Twelve tonebursts were presented each minute; responses to each presentation areshown as horizontally aligned squares. Tone bursts were presented for 1–2min before kynurenic acid was applied, and the responses are shown asthe columns of squares at the top of each record. The times at which theiontophoresis of kynurenic acid was initiated and terminated are indi-cated by dashed horizontal lines. A, Iontophoretic application of kynurenicacid for 5 min (hatched vertical bar) blocked excitation at sites distant fromthe recording electrode. The tip of the recording electrode was 275 mmfrom the ejection barrels. Within 1 min, kynurenic acid completely sup-pressed discharges at the recording electrode. The neuron recovered in 5min and responded to the BF tones bursts as it did before drug applica-tion. Ejection current was 200 nA. B, In this example, the tip of therecording electrode was 100 mm from the ejection barrels, and the neuronwas inactivated twice. For the first inactivation (1), the ejection current ofkynurenic acid was 100 nA applied for 2 min. The neuron recovered after2 min. The neuron was then inactivated a second time (2) with an ejectioncurrent of 200 nA. The higher ejection current suppressed the dischargesand resulted in a longer recovery period. The recordings in both A and Bwere conducted in the same animal.


Thus, the inhibition persisted for 60 msec beyond the duration ofthe 10 msec tone that evoked it. That the inhibition occurred inthe DNLL cell was confirmed by its elimination when bicucullineand strychnine were applied iontophoretically. The maximal pe-riods of persistent inhibition varied among the 50 DNLL neuronsthat we tested and were typically ,60 msec. The average periodof the maximum persistent inhibition was 18 msec and rangedfrom 3 to 80 msec.

Persistent inhibition in the DNLL was evoked not only by tonebursts but also by brief FM sweeps, so long as the FM signal sweptthrough the best frequency of the DNLL cells. The averagepersistent inhibition evoked by FM signals, measured in 21DNLL neurons, was 13 msec and ranged from 4 to 38 msec. In 8of the 21 neurons, we measured persistent inhibition evoked byboth tone bursts and FM sweeps. As illustrated by the neuron inFigure 4, the two types of stimuli evoked inhibitory persistence ofsimilar, but not identical, duration. Tone bursts usually caused a

slightly longer persistent inhibition than did FM sweeps. In theseeight neurons, the average FM-evoked inhibitory persistence was12 msec, whereas the average tone-evoked inhibitory persistencewas 17 msec. Previous studies reported comparable inhibitoryperiods evoked by tones and other signals in DNLL neurons ofmustache bats (Yang and Pollak, 1998).

Discharges evoked by contralateral stimulation aresuppressed during periods of persistent inhibitionThe persistence of inhibition suggests that a signal presented tothe ipsilateral (inhibitory) ear should prevent DNLL neuronsfrom responding to signals presented to the contralateral (excita-tory) ear for a period of time after the inhibitory signal. To testthis, we presented binaural signals in which the excitatory signalswere presented at various times after the inhibitory signals. Anexample is shown in Figure 5. When the signals were presentedsimultaneously, the discharges evoked by stimulation of the exci-tatory ear were completely inhibited (data not shown). As theexcitatory signal was delayed, the discharges continued to besuppressed. In this neuron, discharges began to appear at a delayof 10 msec, and full recovery occurred when the excitatory signalwas presented 30 msec after the end of the inhibitory signal. Thisshows that the persistent inhibition generated by signals at theipsilateral ear suppressed the excitation evoked by signals at thecontralateral ear. The suppression of contralaterally evoked dis-charges during periods of persistent inhibition was seen in all 19neurons tested in Mexican free-tailed bats. Moreover, it was seenpreviously in almost all neurons tested in the mustache batDNLL, where it was also shown that the suppression of contralat-erally evoked discharges during periods of persistent inhibitioncould be rescued by blocking inhibition at the DNLL with bicu-culline and strychnine (Yang and Pollak, 1994c, 1998).

Figure 3. Excitation and inhibition in a DNLL neuron. A, Sustainedresponse evoked by a BF tone burst (20 dB SPL; horizontal bar) presentedto the ear contralateral to the DNLL is shown. B, Background activityevoked by iontophoretic application of glutamic and aspartic acids (Glu/Asp) is shown. C, A 10 msec BF tone burst (40 dB SPL) at the earipsilateral to the DNLL (inhibitory ear) inhibits Glu/Asp-evoked back-ground activity. The inhibition, seen as the gap in the background activity,had a total duration of 70 msec. Thus, the inhibition persisted for 60 msecbeyond the duration of the 10 msec tone burst that evoked the inhibition.D, Ipsilaterally evoked inhibition was abolished when a cocktail of bicu-culline and strychnine was applied. The blockage of inhibition combinedwith the enhanced excitability caused by the Glu/Asp unmasked a sub-threshold excitation. The excitation was not evoked in the absence ofGlu/Asp, even when inhibition was blocked by bicuculline and strychnine.Not all DNLL cells displayed such a subthreshold excitation under theseconditions. Ejection currents were as follows: Glu/Asp, 15 nA, and bicu-culline and strychnine cocktail, 30 nA. Time bars under responses havebeen shifted to illustrate the relationship between the stimulus and re-sponse durations. BF was 35.2 kHz. Bic, Bicuculline; Strych, strychnine.

Figure 4. Persistent inhibition in the DNLL is evoked both by tonebursts and by FM sweeps. Top, Control showing background dischargesevoked by iontophoresis of glutamic and aspartic acid when no stimuluswas presented. Bottom, The gaps in the background evoked by 2 msec tonebursts or 2 msec FM sweeps. The tone burst evoked a total inhibitoryperiod of 16 msec. The initial 2 msec of inhibition occurred over theduration of the tone burst, and the inhibition then persisted for anadditional 14 msec. The 2 msec FM sweep evoked a persistent inhibitionof 12 msec in the same DNLL neuron. The threshold at BF was 210 dBSPL. Time bars under responses have been shifted to illustrate the rela-tionship between the stimulus and response durations. Tone bursts were40.4 kHz (BF) at 30 dB SPL. The FM was 30 dB SPL, the same intensityas the tone burst, and swept from 50 to 30 kHz and thus through the BFof the neuron.


Inactivation of DNLL relieves inhibition in manyICc cellsThe previous section dealt with stimuli that inhibited DNLLcells. In this section we deal with excitation of the DNLL andshow that DNLL excitation provides inhibition to most, but notall, ICc neurons. We studied the effects of DNLL excitation in 78ICc neurons by evaluating their EI properties before, during, andafter DNLL inactivation produced by iontophoretic applicationof kynurenic acid. In 37% (29 of 78) of the ICc cells, inactivatingthe DNLL had no effect on their EI properties. In these neurons,the inhibition evoked by increasing the signal intensity to theipsilateral ear was virtually the same during DNLL inactivationas it was before DNLL inactivation (Fig. 6). This suggests thatthe DNLL played little or no role in the formation of the EIproperties in these ICc cells.

In most ICc neurons (49 of 78; 63%), however, DNLL inacti-vation reduced ipsilateral inhibition by at least 20%, indicatingthat the DNLL played a prominent role in the formation of theirEI properties. On average, the maximum relief from inhibitionamong these 49 ICc cells was 41% and ranged from 20 to 100%(Fig. 7). An example is shown in Figure 8. This was a stronglyinhibited EI neuron in which signal intensities of 30 and 40 dBSPL at the ipsilateral (inhibitory) ear completely suppresseddischarges evoked by contralateral (excitatory) stimulation. Dur-ing inactivation of the opposite DNLL, ipsilateral (inhibitory)

signals produced a much smaller decline in spike count than thesame signals did before inactivation. For example, with an ipsi-lateral intensity of 30 dB SPL, the spike count was 73% of themaximal count, a 27% suppression, whereas before inactivationthe same ipsilateral intensity caused complete suppression. Whenthe intensity at the inhibitory ear was increased to 40 dB SPL,which had also previously suppressed discharges completely, thespike count was reduced to 57% of maximum, a 43% suppression.The DNLL recovered within 10 min, and stimulation at theinhibitory ear again suppressed spike counts by 100%.

As with the cell in Figure 8, DNLL inactivation caused sub-stantial, but not complete, relief from inhibition in most ICc cells.To evaluate whether DNLL inactivation eliminated most, if notall, ipsilaterally evoked GABAergic inhibition, we iontophoreti-cally applied bicuculline to the ICc cell either during DNLLinactivation or after recovery from DNLL inactivation in 18neurons. Bicuculline blocks not only the GABAergic innervationfrom the DNLL but other GABAergic inputs to the ICc cell aswell. In general, relief of inhibition in the 18 cells was greater withbicuculline than with DNLL inactivation. Two representativeexamples are shown in Figure 9. For the neuron in Figure 9A,

Figure 5. Persistent inhibition in DNLL evoked by an inhibitory signalat the ear ipsilateral to the DNLL suppresses discharges evoked byexcitatory signals presented to the contralateral ear. Top, Complete sup-pression of contralateral (excitatory) signals by ipsilateral signals when thetwo were presented at an interval of 0 msec is shown. Bottom, Partialrecovery occurred when the excitatory signal followed the inhibitorysignals by 10–20 msec. Full recovery first occurred 30 msec after theinhibitory signal was over. The excitatory signals were 5 msec BF tonebursts and were 30 dB SPL. The inhibitory signals were 10 msec BF tonebursts at 50 dB SPL. BF was 36.1 kHz.

Figure 6. An ICc neuron that was not influenced by inactivation of theopposite DNLL. Bottom, Raster displays are shown of the responsesevoked by binaural signals in which the intensity at the ear ipsilateral tothe ICc (inhibitory ear) was increased by increments of 5 dB. Top,Suppression is plotted graphically. Here the suppression evoked by increas-ing ipsilateral intensities is plotted as the percentage of maximum responseevoked. The functions obtained before (control) and during DNLL inac-tivation are similar, suggesting that the DNLL had little or no effect on theipsilaterally evoked spike suppression in this cell. BF was 23.3 kHz.


DNLL inactivation reduced inhibition from ;98% of the maxi-mum response before inactivation to ;43% when intensity at theinhibitory ear was 50–60 dB SPL. However, when bicuculline wasiontophoresed while the DNLL was inactivated, the inhibitionwas reduced even further and relieved almost 80% of the inhibi-tion. For the neuron in Figure 9B, DNLL inactivation relieved;32% of the inhibition (at 50 dB SPL). After recovery fromDNLL inactivation, bicuculline was applied and relieved almostall inhibition. Thus DNLL inactivation relieved inhibition, butwith few exceptions, it alone did not eliminate all inhibitionevoked by the ipsilateral (inhibitory) ear. This suggests either thatthere were, in addition to the DNLL, other sources of inhibitionevoked by the inhibitory ear or that iontophoresis of kynurenicacid did not completely block DNLL activity. Although we can-not distinguish between these interpretations, the relevant pointis that DNLL inactivation relieved at least 20% of the inhibitionevoked by the ipsilateral (inhibitory) ear in 63% of the ICcneurons that we tested.

Impact of DNLL persistent inhibition on ICc neuronsHere we test the hypothesis mentioned previously, that the func-tional impact of persistent inhibition in the DNLL on the oppo-site ICc is to influence the processing of multiple sound sourcesthat emanate from different regions of space (Yang and Pollak,1994c, 1998; Pollak, 1997). The hypothesis is shown diagrammat-ically in Figure 10 and logically combines two principal featuresof the circuit. The first feature, shown in Figure 10A, applies to abinaural signal that favors the ipsilateral (inhibitory) ear whenpresented alone. It shows that the excitation evoked by one earcan be strongly or completely suppressed by stimulation of theother ear and that the suppression at the ICc cell derives from theGABAergic inhibition provided by the opposite DNLL. Thesecond feature is that a signal that favors the ear ipsilateral to theDNLL generates a persistent inhibition in DNLL neurons thatinnervate the opposite ICc (Fig. 10B). In Figure 10B we show this

signal as a monaural signal or prepulse, although the same logicwould apply to a binaural signal with an IID that favors the earipsilateral to the DNLL. By combining these features, the hy-pothesis predicts that presenting an initial signal, which is eithermonaural as shown here or binaural with an IID that producesDNLL persistent inhibition, should reconfigure the circuit andthereby change the way the ICc cell responds to a binaural trailingsignal. Specifically, the initial presentation of a monaural prepulse(or a binaural signal) should drive the ICc, but it should alsogenerate a persistent inhibition in the DNLL (Fig. 10B). The keyargument is that during the period when the DNLL is persis-tently inhibited, the ICc cell should be deprived of its ipsilaterallyevoked inhibition, thereby allowing the ICc cell to respond totrailing binaural signals to which it previously responded poorlyor not at all (Fig. 10C). We also emphasize that a corollary of thehypothesis applies to ICc cells that are not innervated by theopposite DNLL. In those cells, the hypothesis predicts that the

Figure 7. Maximum relief from inhibition by DNLL inactivation in 78ICc neurons, the EI properties of which were tested before and duringDNLL inactivation. Normalized spike counts to binaural signals weredetermined before and during DNLL inactivation. The difference be-tween the value before and during inactivation is the percentage relieffrom inhibition. Shown here is the largest percentage relief for each cell.The average maximum relief was 41% among the 49 neurons that hadrelief of 20% or greater.

Figure 8. DNLL inactivation relieved ipsilaterally evoked spike suppres-sion in an ICc neuron. Bottom, Raster displays are shown of spikesuppression evoked by increasing intensity at the ear ipsilateral to the ICc(inhibitory ear) before (control), during, and after DNLL inactivation(recovery). The relief from inhibition caused by DNLL inactivation isespecially apparent at ipsilateral intensities of 30–40 dB SPL. Top, Thepercentage of maximum response evoked by increasing ipsilateral inten-sities before and during DNLL inactivation and when the neuron recov-ered from inactivation is graphically plotted. BF was 29.5 kHz.


introduction of a prepulse should have no effect on the trailingbinaural signal; i.e., the ICc cell should respond to the trailingsignals as it did when the same trailing signals were presentedalone.

To test this hypothesis, we recorded from 96 EI neurons in theICc. In each neuron we presented a monaural signal, or prepulse,to the ear that both excited the ICc and generated a persistentinhibition in the DNLL. The prepulse was followed by binauralsignals with different IIDs (Fig. 10C). We could not evaluate theeffects of an initial signal (prepulse) in 42 of the 96 cells. In thosecells, recovery was poor, and trailing signals evoked either noresponses or very weak responses when they followed the firstpulses by 15 msec or less. In these cases the prepulse presented tothe contralateral ear apparently evoked both an excitation fol-lowed by a long-lasting inhibition at the ICc cell, which sup-pressed responses to the trailing signals for $15 msec. Longrecovery times have been reported in previous IC studies con-cerned with the processing of multiple sound sources (Yin, 1994;Fitzpatrick et al., 1995). The functional significance of this con-tralaterally evoked persistent inhibition exhibited by many ICcneurons is unknown, and this puzzling feature of the IC has beendiscussed in a previous report (Bauer et al., 2000).

Of the 96 cells, 54 cells (56%) recovered quickly. As shownbelow, almost all of these neurons were consistent with thehypotheses presented above for ICc cells that are innervated bythe DNLL and for ICc cells that are not. We turn first to 23 of the54 ICc cells in which a monaural prepulse had no effect on the

Figure 9. Two ICc neurons in which DNLL inactivation partially re-lieved ipsilaterally evoked inhibition but blocking GABAergic inhibitionwith bicuculline produced even greater relief. Signals were 10 msec tonebursts at the BF. BFs were 27.1 kHz (A) and 24.9 kHz (B). The ejectioncurrent for bicuculline was 60 nA in both A and B. BIC, Bicuculline.

Figure 10. Schematic diagram of auditory pathways showing how aprepulse presented to the ear contralateral to the ICc could reconfigurethe circuit and allow the ICc cell to respond to a trailing signal to whichit was unresponsive when presented alone. A, A binaural signal with anIID that favors the ear ipsilateral to the ICc drives two projections. Thefirst is an excitatory projection from a lower monaural nucleus (e.g., thecochlear nucleus) and a GABAergic inhibitory projection from the oppo-site DNLL. At this IID, the inhibitory projection from the DNLLsuppresses the excitation from the contralateral ear. B, A monauralprepulse evokes a persistent inhibition in the ipsilateral DNLL (indicatedby the stippling) and excites the contralateral ICc. Inhibition in the DNLLis evoked by a glycinergic projection from the ipsilateral LSO and aGABAergic inhibition from the opposite DNLL through the commissureof Probst (Yang and Pollak, 1994a,b). The excitation of the ICc is via anexcitatory projection from a lower monaural nucleus, shown here gener-ically as coming from the cochlear nucleus. C, The initial presentation ofthe monaural prepulse persistently inhibits the DNLL but excites the ICc,in the same way as shown in B. When a trailing binaural signal that favorsthe inhibitory ear follows shortly thereafter, the ICc neuron now respondsto the trailing signal. The reason is that the prepulse generated a persis-tent inhibition in the DNLL that deprived the ICc cell of the inhibitionthat would be evoked by a binaural trailing signal if it were presentedalone. Thus, the weaker stimulus at the contralateral (excitatory) earevoked by the trailing signal is now free to drive the ICc cell.


responses to the trailing binaural signal. The DNLL was inacti-vated while recording from 12 cells. In three of these cells, DNLLinactivation relieved ipsilateral inhibition although the prepulsedid not. However, in 9 of 12 cells (75%), neither a prepulse norDNLL inactivation relieved inhibition, and thus neither of themanipulations affected the EI properties evoked by the trailingbinaural signals. Stated differently, these neurons responded tothe trailing binaural signal as they did when same binaural signal(the trailing signal) was presented alone. A representative exam-ple is shown in Figure 11. This neuron apparently was notinnervated by the opposite DNLL, and its EI property may havebeen created in the LSO and then imposed on the ICc via thecircuit shown in Figure 1B.

In marked contrast were 31 cells in which a monaural prepulsesubstantially relieved ipsilateral inhibition. An example is shownin Figure 12. When a binaural signal was presented without aprepulse, the discharges evoked by the contralateral ear wereinhibited by ipsilateral intensities of 40 dB SPL or greater (Fig. 12,bottom lef t). When a monaural prepulse was introduced and thesame binaural signals followed the prepulse 1.0 msec later, therewas only weak inhibition of the binaural signal, even at an ipsi-lateral intensity of 45 dB SPL, an intensity that completely inhib-ited the same binaural signal when there was no prepulse. Therelief from inhibition afforded by the prepulse declined as theinterval between the prepulse and trailing signals was lengthened(Fig. 12, top). When the interval was lengthened to 3 msec, theamount of relief was slightly reduced, and when the delay waslengthened further, to 5 msec, the prepulse no longer producedany relief from inhibition. Thus, the presentation of the prepulseat appropriate intervals relieved inhibition, allowing the ICcneuron to respond to the binaural trailing signal at IIDs thatcompletely inhibited the cell when the trailing signal was pre-sented alone. The intervals that were effective for relieving inhi-bition were very short in this ICc neuron. Our interpretation isthat this ICc cell received innervation from DNLL cells that hadvery short periods of persistent inhibition. Because periods ofpersistent inhibition were longer in most DNLL cells, the effec-tive intervals between the prepulse and trailing signals were alsosubstantially longer in other ICc neurons, as we show below.

Although we did not inactivate the DNLL in the neuron shownabove, we did inactivate the DNLL with kynurenic acid in 11cells in which a prepulse relieved the inhibition produced by thetrailing binaural signal. In all 11 cells, relief from inhibition wasobtained from DNLL inactivation, and the relief was similar tothe relief obtained with a prepulse. An example is shown inFigure 13. Here we first selected an IID that produced a strong,but not complete, suppression of contralaterally evoked dis-charges and measured the relief from inhibition against thatsuppression. In this cell, the prepulse was an FM sweep that didnot drive the ICc cell, although it swept through the BF of the ICccells. The trailing binaural signals were BF tone bursts. Becausewe have shown that FM sweeps effectively generate persistent

Figure 11. An ICc cell in which the responses evoked by a trailingbinaural signal were not influenced either by a monaural prepulse or byDNLL inactivation. The percent-normalized responses for the prepulseand DNLL inactivation conditions are for the trailing binaural signals.Both prepulse and trailing binaural signals were 10 msec FM signals thatswept from 40 to 20 kHz. The FM prepulse was 40 dB SPL.

Figure 12. Prepulses relieved ipsilateral inhibition evoked by trailingbinaural signals. Bottom lef t, Raster displays of binaural signals presentedalone. Note the suppression of responses evoked by contralateral stimu-lation as the intensity at the ear ipsilateral to the ICc was increased.Bottom middle, right, Raster displays evoked by an FM prepulse at the earcontralateral to the ICc, followed 1.0 and 5.0 msec later by trailingbinaural FM signals. Note the relief from inhibition afforded by the 1.0msec interval when the ipsilateral intensities of the trailing signals were40–45 dB SPL. Relief was over at the 5.0 msec interval, presumablybecause the persistent inhibition in the DNLL had ended at that time.FM signals swept from 40 to 15 kHz. Top, Suppression produced byipsilateral intensities as a percentage of the maximum response. Includedare data from the 3.0 msec interval between the prepulse and trailingsignals, which are not shown in raster displays.


inhibition in the DNLL, we assume that if the DNLL innervatedthis ICc cell, the FM prepulse should inhibit those DNLL cellsalthough the FM sweep did not drive the ICc cell. Figure 13Ashows that a 15 dB SPL tone burst presented only to the excitatoryear evoked 39 spikes, but when the same excitatory signal waspresented simultaneously with a 25 dB SPL signal at the inhibi-tory ear, only six spikes were evoked. When the DNLL wasinactivated with kynurenic acid, the same binaural signal that

evoked previously only six spikes now evoked 30 spikes, showingthat the opposite DNLL provided most, if not all, of the ipsilat-eral inhibition evoked by the binaural signal. Introduction of theFM prepulse relieved the inhibition substantially. At an intervalof 10 msec, the trailing binaural signal evoked 26 spikes (Fig.13B), whereas the same binaural signal presented alone evokedonly six spikes. This amount of relief from inhibition was similarto the relief that occurred when the DNLL was pharmacologi-cally inactivated. The amount of relief declined as the intervalwas lengthened to 20 msec (trailing signal evoked 17 spikes), andat an interval of 30 msec the trailing signal evoked only ninespikes, a spike count comparable with the six spikes obtainedwhen the binaural trailing signal was presented without the pre-pulse. Thus, as with the cell in Figure 12, the prepulse not onlyrelieved inhibition but its efficacy dissipated over time, as did thepersistent inhibition in the DNLL (e.g., Fig. 5). Moreover, be-cause most, if not all, of the ipsilateral inhibition in this cellderived from the opposite DNLL, the relief from inhibitionafforded by the prepulse must have been caused by the generationof persistent inhibition in the DNLL by the prepulse.

Initial binaural signals also relieve inhibition generatedby trailing signals in the ICcIn the above sections we showed that a monaural signal relievedthe inhibition generated by a trailing binaural signal. Those con-ditions were artificial in that sounds almost always stimulate bothears. Here we show that relief from inhibition was also producedwith two binaural signals, in which the first had an IID thatfavored the contralateral ear and therefore drove the ICc cell andinhibited the DNLL, whereas the second, or trailing signal, hadan IID that favored the ipsilateral ear, which drove the DNLLand inhibited the ICc cell. This stimulus arrangement simulatesthe reception of two signals that emanate from different regionsof space. As with the results shown in the previous section, therelief from inhibition was obtained in ICc cells that were inner-vated by the DNLL, and no relief was observed in ICc cells thatwere not innervated by the DNLL.

We tested 12 ICc cells with two binaural signals. Two of the 12cells showed no inhibitory relief from the initial signal, and inboth of those cells DNLL inactivation also provided no relief. In10 cells, however, initial signals relieved inhibition evoked by thetrailing signals. In four of those cells, we also inactivated theDNLL with kynurenic acid. In all four neurons, the reliefachieved with the initial binaural signal was mimicked by phar-macological inactivation of the DNLL. As with the neurons inFigures 12 and 13, we also show that the relief afforded by theinitial binaural signal is effective only for a limited time windowthat presumably corresponds to the periods of persistent inhibi-tion in the DNLL.

We illustrate relief of inhibition with the two ICc cells inFigures 14 and 15. For the neuron in Figure 14, the IID of thetrailing signal was 0 dB, and responses to this binaural signalpresented alone are shown in the top panel. The IID of the initialsignal was 120 dB (excitatory ear more intense). When the initialand trailing signals were presented at intervals ranging from 2 to20 msec, the spike counts evoked by the trailing binaural signalwere much larger than were the spike counts evoked by the samebinaural signal when it was presented alone. The enhanced spikecounts at the ICc evoked by the trailing signal were presumably aconsequence of the persistent inhibition at the DNLL that wasgenerated by the initial binaural signal and that deprived the ICccell its inhibitory input from the ipsilateral ear. The strength of

Figure 13. DNLL inactivation mimicked the relief of inhibition pro-duced by a monaural prepulse. A, Control responses evoked without aprepulse. Signals presented to the ear contralateral to the ICc (theexcitatory ear) are shown as unfilled bars, whereas signals presented to theear ipsilateral to the ICc (the inhibitory ear) are shown as hatched bars.BF tone bursts (30 kHz) presented to only the contralateral (excitatory)ear at 15 dB SPL evoked 39 spikes. Binaural tone bursts, having the samecontralateral intensity (15 dB SPL) and an ipsilateral intensity of 25 dBSPL, evoked only six discharges, an 85% suppression. When the DNLLwas inactivated, the same binaural signal evoked 30 spikes, a 23% sup-pression. Thus, inactivating the DNLL relieved ;62% of the ipsilaterallyevoked inhibition. The diagonal slash marks on the horizontal line indicatethat tone alone, binaural, and DNLL inactivation records were eachobtained separately and independently. B, Relief from inhibition affordedby a 5.0 msec FM prepulse presented to the ear contralateral to the ICc(the excitatory ear). The FM swept from 40 to 20 kHz. The neuron wasunresponsive to the FM signal but responded briskly to the trailingbinaural tone bursts. The trailing binaural signal evoked 26 spikes when itfollowed the prepulse by 10 msec (a 33% suppression). Thus the prepulserelieved ;52% of the ipsilaterally evoked inhibition, a value close to the62% relieved by DNLL inactivation. Less relief was achieved when theinterval was lengthened to 20 msec, and there was no relief at 30 msec, thelongest interval.


the persistent inhibition at the DNLL was greatest during thetimes immediately after the initial signal and declined over time,as shown previously. Thus, the highest spike count was evoked atthe shortest delay (2 msec) and then declined as the delay waslengthened to 10 and 20 msec. At the longest delays, of 30 and 40msec, the spike counts evoked by the trailing signal were compa-rable with the spike counts evoked by the same signal whenpresented alone. It appears, therefore, that the persistent inhibi-tion at the DNLL was over at the longest intervals, therebyallowing the ipsilateral tone of the trailing signal to again inhibitthe discharges evoked by the contralateral tone.

A similar result is shown for the neuron in Figure 15. Here thefirst signal relieved most of the ipsilateral inhibition at delays of 3

and 13 msec, and relief was over when the interval was 23 msec.As shown in Figure 15A, inactivation of the DNLL withkynurenic acid eliminated all of the ipsilateral inhibition, suggest-ing that GABAergic innervation from the DNLL could accountfor all of the ipsilateral inhibition at this ICc cell.

The time periods over which relief from inhibition dissipatedvaried among the cells to which we presented first and trailingsignals at a variety of delays. In some ICc cells, the relief affordedby the first signal was over at short delays (e.g., Fig. 12), whereasin other neurons the relief lasted for .15 msec (e.g., Fig. 14). We

Figure 14. Initial binaural signals that favor the ear contralateral to theICc (the excitatory ear) relieve ipsilateral inhibition evoked by trailingsignals. All signals were 7.0 msec FM signals that swept from 45 to 20 kHz.Signals presented to the ear contralateral to the ICc (the excitatory ear)are shown as unfilled bars, whereas signals presented to the ear ipsilateralto the ICc (the inhibitory ear) are shown as hatched bars. Top, Controlshowing that 13 spikes were evoked by binaural signals presented withequal intensities at the two ears (IID, 0 dB). Bottom, An initial binauralsignal, having an IID of 120 dB (excitatory ear louder), relieved ipsilat-eral inhibition evoked by the trailing binaural signal. When the intervalwas 2.0 msec, the trailing signal evoked 47 spikes, whereas it evoked onlysix spikes when presented alone. Increasing the interval between theprepulse and trailing signal caused progressively less relief. Relief wasover at 30 msec, because the trailing signal evoked almost the same spikecount as it did when presented alone. C, Contralateral; I, ipsilateral.

Figure 15. DNLL inactivation mimicked the relief of inhibition pro-duced by an initial binaural signal. All signals were 10 msec FMs thatswept from 40 to 20 kHz. Signals presented to the ear contralateral to theICc (the excitatory ear) are shown as unfilled bars, whereas signalspresented to the ear ipsilateral to the ICc (the inhibitory ear) are shownas hatched bars. A, Control responses evoked without a prepulse. Tonebursts presented to only the contralateral (excitatory) ear at 20 dB SPLevoked 32 spikes. A binaural signal, having the same contralateral inten-sity (20 dB SPL) but an ipsilateral intensity of 40 dB SPL, evoked only sixdischarges, an 82% suppression. When the DNLL was inactivated, thesame binaural signal evoked no suppression. Thus inactivating the DNLLrelieved 100% of the ipsilaterally evoked inhibition. B, Relief from inhi-bition afforded by a binaural FM prepulse. The IID of the prepulse was110 dB, and the excitatory ear was more intense. The trailing binauralsignal evoked 26 spikes when it followed the prepulse by 3.0 msec (asuppression of 19%). Thus the prepulse relieved ;81% of the ipsilaterallyevoked inhibition. Slightly less relief was achieved when the interval waslengthened to 13 msec, and relief was over when the interval was 23 msec.


obtained an estimate for how long, after an initial signal, thepopulation of ICc neurons might be relieved. To do this, in eachneuron we measured the percentage relief at the shortest delay,which almost always produced the greatest relief, and the firstdelay at which relief was no longer present. On average, the reliefdissipated by 9%/ms. Thus, on average, relief from inhibitionattributable to an initial signal was over after ;10 msec.

DISCUSSIONHere we showed that the DNLL provides a potent GABAergicinhibition to the opposite ICc. The inhibition shapes the EIproperties of most ICc cells, a finding consistent with numerousprevious studies (Faingold et al., 1991, 1993; Li and Kelly, 1992;Park and Pollak, 1993, 1994; Klug et al., 1995; Kidd and Kelly,1996; van Adel et al., 1999; Kelly and Kidd, 2000). One EIproperty shaped in the ICc is the IID at which the cell ismaximally inhibited. For convenience, we refer to this IID as theIID sensitivity of the neuron. Because of DNLL influences, IIDsensitivities among the ICc population range from approximately230 dB (ipsilateral ear more intense) to approximately 110 dB(contralateral ear more intense) (Wenstrup et al., 1988a; Park andPollak, 1993; Park, 1998).

The significance of this distribution of IID sensitivities is thatit provides the substrate for the population coding of IIDs gen-erated by high-frequency sounds (Fuzessery and Pollak, 1984;Fuzessery et al., 1985; Wenstrup et al., 1986, 1988b). Each IIDsensitivity of each neuron determines its spatial receptive field,the regions of space from which sounds evoke discharges (Fuz-essery and Pollak, 1984, 1985; Wenstrup et al., 1988b). Assumingthat a wide range of IID sensitivities is represented in isofre-quency contours of the ICc, as occurs in the mustache bat (Wen-strup et al., 1986), the arrangement translates into a representa-tion of the IID generated by the frequency to which the neuronsin that contour are tuned. The idea is that when a complex soundemanates from a location in space, each frequency in the spec-trum of the sound generates a particular IID. The IID generatedby each frequency is then coded by the EI population response inthe corresponding frequency contour, where some EI neuronswith particular IID sensitivities are inhibited whereas other neu-rons, with different IID sensitivities, are excited. Changing thelocation of the sound changes the IID generated by each fre-quency and, thus, the population response in each frequencycontour. In this way, the EI populations in the frequency contoursof the ICc can collectively encode the location of a sound sourcein both azimuth and elevation from most locations in the frontalhemifield (Fuzessery and Pollak, 1984, 1985; Pollak et al., 1986).

The importance of collicular EI neurons for sound localizationis illustrated both by the disruption in localization acuity causedby lesions of the DNLL and by sectioning of the commissure ofProbst (Ito et al., 1996; Kelly et al., 1996). The DNLL, however,not only shapes the IID sensitivities of the ICc population, but italso changes the IID sensitivities of many ICc cells to trailingsounds. With only a single sound source, all ICc neurons with agiven IID sensitivity respond to a frequency from a particularlocation in the same way, regardless of whether or not their IIDsensitivity is shaped by the DNLL. However, an initial signal thatpersistently inhibits the DNLL will degrade the spatial selectivityof those ICc cells for a trailing signal by expanding the regions ofspace from which the trailing sound can drive those cells. Ourresults show that this general rule is applicable to most EI col-licular neurons that are innervated by the opposite DNLL.

The persistent inhibition in the DNLL may haveconsequences for other aspects ofbinaural processingThe effects of DNLL persistent inhibition on the ICc should beespecially significant for those signals that generate IIDs thatchange over time, such as moving sound sources or multiplesounds that emanate from different regions of space. That theDNLL may influence motion sensitivity of ICc cells is suggestedby the differential influence that positive and negative IIDs ofinitial sounds have on the responses to subsequent sounds. Thesefeatures suggest that a moving sound source that begins in thecontralateral sound field and moves with a certain velocity intothe ipsilateral sound field should evoke more vigorous responsesin an ICc cell than would be evoked when the sound sourcemoved from the ipsilateral into the contralateral sound field. Thistype of sensitivity for the direction of movement has been re-ported for neurons in the IC of several mammals, including bats(Spitzer and Semple, 1993; Kleiser and Schuller, 1995; Wilsonand O’Neill, 1998; McAlpine et al., 2000). Moreover, Keller andTakahashi (1996) proposed a similar mechanism to explain direc-tionally selective motion sensitivity in IC neurons of the barn owl.

The demonstration that an initial signal can change the spatialselectivity of ICc cells to a trailing signal suggests that the DNLLcircuitry could contribute to a precedence-like effect. The prece-dence effect was discovered in human psychophysical studies andis caused by a mechanism that suppresses the directional infor-mation carried by echoes. It explains how, in a reverberant room,a listener can hear only a single sound and not the sequence ofseparate sounds produced by the echoes reflected from the vari-ous surfaces and objects in the room (Wallach et al., 1949;Blauert, 1983; Zurek, 1987; Litovsky et al., 1999).

Precedence is classically demonstrated with two speakers, sep-arated along the same plane in space (Wallach et al., 1949;Litovsky et al., 1999). The speakers emit identical sounds, but thesound from one speaker is presented a few milliseconds beforethe sound from the other speaker. Under these conditions, normallisteners hear a single composite sound and perceive the compos-ite sound as originating from the leading speaker. The secondsound is heard by the listener but is not perceived as a separatesound, nor does it influence the perceived location of the com-posite sound. Rather the second sound fuses with the first soundand contributes to the overall volume and timbre of the fusedsound. Whether the listener hears a single, fused sound or twoseparate sounds depends on the delay between the two sounds, aswell as on the duration and the complexity of the sound. If theinterval between the first and second sounds exceeds an upperlimit, the two sounds are no longer heard as a single sound but astwo separate sounds in succession, each with a perceived locationin space.

One significant feature of precedence, that a trailing sound isheard but not localized, correlates closely with the DNLL influ-ences that we observed. Specifically, persistent inhibition inDNLL evoked by the first sound does not suppress the activity ofICc neurons to the trailing sound. Rather, it allows many ICc cellsto fire to trailing signals to which they were previously poorlyresponsive or unresponsive, thereby expanding the regions ofspace from which sound could drive those ICc cells. Thus, theactivity in the ICc should allow the animal to hear trailing sounds,but DNLL innervated cells should also degrade the accuracy withwhich the ICc population codes for their locations because thesecells constitute a large proportion of the EI population in the IC.


The degradation of the population code, in turn, should impairthe localization of the trailing signal.

Another feature of agreement is the time course of precedenceand the time course of relief from inhibition at the ICc affordedby an initial signal. Precedence persists from a few to 20–40 msec,depending on the experiment and type of stimulus. The averageperiod during which a first signal relieved the inhibition evoked bya trailing signal in the ICc is ;1–20 msec, which falls between theshort and long periods estimated in psychophysical experiments(Blauert, 1983; Litovsky et al., 1999).

We point out that the mechanisms we showed here can onlyaccount for the precedence of high-frequency sounds, which areinitially processed binaurally in the LSO. Moreover, the firstsound has to emanate from a location off the midline, whichgenerates IIDs that evoke a persistent inhibition in the DNLL,followed by sounds that emanate from the other acoustic hemi-field. If the first sounds are located at or around the midline andproduce IIDs at or around 0 dB, persistent inhibition would notbe generated in the DNLL, and thus the spatial selectivity of ICccells for trailing sounds would not be degraded.

Precedence, however, also occurs with low frequencies, whichare initially processed binaurally in the medial superior olive.Furthermore, precedence is not limited to initial and trailingsounds that emanate from opposite regions of the frontal soundfield. These features, together with the results from previousstudies (Wickesberg and Oertel, 1990; Yin, 1994; Fitzpatrick etal., 1995; Litovsky and Yin, 1998a,b), suggest that precedence isalmost certainly the consequence of several mechanisms that actat a variety of levels along the auditory pathway and that the relieffrom DNLL inhibition at the ICc shown here may be but one ofthose mechanisms.

Precedence occurs in many animalsMechanisms that suppress the localization of reverberations mustbe under strong selective pressures because precedence is awidespread, if not universal, feature of auditory systems. Prece-dence has been found in insects (Wyttenbach and Hoy, 1993), birds(Keller and Takahashi, 1996), and a variety of mammals includingrodents (Kelly, 1974; Wickesberg and Oertel, 1990), carnivores(Cranford and Oberholtzer, 1976; Yin, 1994; Litovsky and Yin,1998a,b), rabbits (Fitzpatrick et al., 1995), and humans (Litovsky etal., 1999). We suggest that bats should be added to this list.

Bats have to cope extensively with acoustic reverberations intheir daily lives. The Mexican free-tailed bats used in this study,for example, live in caves where they congregate in large coloniesthat often number in the millions. They use a rich repertoire ofcommunication calls that they use for a wide variety of socialinteractions (Balcombe, 1990; Balcombe and McCracken, 1992;French and Lollar, 2000). Caves are highly reverberant, and inthis environment precedence would facilitate the perception ofcommunication signals from other bats by allowing the bat tolocalize the first sound received. Precedence would thus preventthe bat from perceiving each reverberation as a separate soundwith its own location, similar to the way that precedence inhumans prevents the potential confusion from the multiple re-flections in a reverberant room. Precedence might also be advan-tageous for echolocation. While flying either in caves or amongthe vegetation outside, orientation calls are emitted that are re-flected as echoes from objects ahead. The sound reflected from agiven object not only reflects directly back to the bat as a primaryecho but it also reflects or scatters in a variety of directions. Thesound scattered from the first object may then be reflected again,

but now from other objects, producing secondary, weaker echoesthat are also reflected back to the bat. Similar to the argument forcommunication sounds, precedence could be advantageous in al-lowing the bat to localize and focus on primary objects without theconfusion that would occur if each of the secondary echoes wereperceived as separate targets with their own locations.

Concluding commentsHere we showed that both the excitation and persistent inhibitionin the DNLL are key factors that influence binaural processing inthe ICc. The significance of the DNLL innervation is to create anemergent property in the ICc, a property that is not possessed byLSO neurons or by IC cells that are not innervated by the DNLL.That property is a change in the binaural responsiveness of theICc cell, a change produced by the reception of an earlier soundthe IID of which is strongly excitatory to the ICc cell. Thesefeatures suggest that the circuitry linking the DNLL with thecontralateral ICc is important for the processing of signals theIIDs of which change over time, such as the IIDs that would begenerated by moving stimuli or by multiple sound sources thatemanate from different regions of space.

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